Despite extensive diabetes related research, the sequence of events which occur subsequent to the interaction of insulin with the cell membrane and the way this signal is coupled to the cellular machinery are not understood. The particular area which this proposal addresses is the mechanism by which the antilipolytic effect of insulin is mediated in adiopocytes. A major obstacle in this field, as well as in elucidating the mechanism of insulin action in general, is the lack of an adequate insulin-responsive broken cell system. In this proposal this technical limitation is overcome through the use of a unique digitonin permeabilized adipocyte preparation which, despite loss of its cytosolic contents, has an intact lipolytic pathway which responds to low MW effectors (cAMP, ATP, adenosine) as well as physiological concentrations of insulin. Thus, it embodies the best characteristics of intact and broken cell preparations. Toward the ultimate goal of explaining the mechanism of insulin's antilipolytic effects, this proposal sets four specific objectives: 1) to further characterize the antilipolytic effect of insulin on lipolysis in the digitonin-permeabilized adipocytes; 2) to characterize with "in situ" assays the effects of insulin on adenylate cyclase, low Km cyclic AMP phosphodiesterase and cyclic AMP dependent protein kinase, three enzymes that are strong candidates for mediating the antilipolytic effect of insulin; 3) to characterize the effect of insulin on intracellular protein phosporylation, specifically that of the hormone-sensitive lipase, in the permeabilized cells and to correlate phosphorylation with lipolytic activity; and 4) examine the insulin-like characteristics of several putative intracellular insulin mediators (i.e., H202, adenosine, insulin-generated chemical mediator) on the processes described in 1 through 3. By examining the effects of insulin on the overall lipolytic pathway and on several key enzymes in the pathway while maintaining comparable assay conditions, conclusions can be drawn about the relative contributions of these sites to antilipolysis. Additionally, by explaining the mechanism by which insulin mediates this process, the knowledge obtained may be applicable to the understanding of other processes affected by insulin.